Vibrio cholerae is the cause of cholera and a category B agent. Protective immunity to V. cholerae is poorly understood, and development of highly effective cholera vaccines has been problematic. We have preliminary evidence that following wild type cholera, immune responses are prominent against the main subunit of toxin co-regulated pilus (TcpA), a V. cholerae virulence factor required for intestinal colonization of humans. TcpA is expressed in vivo during infection; parenteral and oral killed cholera vaccines do not contain TcpA, and parenteral and killed oral cholera vaccines are only moderately protective against cholera for relatively short periods of time (30-80%; 3-6 months). In addition, we have preliminary evidence that anti-TcpA responses are not prominent following oral immunization with live attenuated cholera vaccines (such as Peru15), despite induction of prominent vibriocidal immune responses following vaccination. We also have preliminary evidence that transcutaneous immunization with TcpA is highly immunogenic, and that combination immunization that includes mucosal (oral) vaccination with live attenuated cholera vaccines followed by transcutaneous boosting with purified antigen induces very robust mucosal and systemic immune responses. We hypothesize that oral immunization with Peru15 and transcutaneous boosting with purified TcpA would induce prominent and protective mucosal and systemic immune responses that more closely mirror what occurs after wild type cholera. In this revised R21 developmental project, we have two Specific aims: (1) To further investigate the potential of transcutaneously applied TcpA to act as a protective immunogen against cholera in a mouse model (evaluating issues of dosing, timing, interval, and safety). (2) To investigate combination immunization of oral priming with Peru15 and transcutaneous boosting with purified TcpA in mice, measuring systemic and mucosal immune responses and protection against cholera in the mouse challenge model. This project would provide pivotal preliminary information on whether induction of anti-TcpA responses would complement immune responses induced by a currently available cholera vaccine, and whether mucosal-transcutaneous combination immunization strategies can induce prominent mucosal and systemic immune responses protective against a mucosal pathogen. Data generated by this developmental R21 project would lay the foundation for a more detailed mechanistic immunological analysis of combination immunization, as well as facilitate subsequent evaluation in humans, and could possibly lay the foundation for a new paradigm of combination immunization regimens against mucosal pathogens. Relevance: Vibrio cholerae is the cause of cholera and a category B agent. This project would investigate whether mucosal-transcutaneous combination immunization strategies against cholera can induce prominent mucosal and systemic immune responses protective against a mucosal pathogen. Data generated by this developmental R21 project would lay the foundation for a more detailed mechanistic immunological analysis of combination immunization, as well as facilitate subsequent evaluation in humans, and could possibly lay the foundation for a new paradigm of combination immunization regimens against mucosal pathogens. [unreadable] [unreadable] [unreadable]